This invention is directed to plant regeneration and transformation of cotton, particularly cotton of the species Gossypium hirsutum L.
In recent years many tissues of diverse origin from plants belonging to different taxonomic groups have been established as in vitro tissue culture. Some of the factors controlling growth and differentiation of such cultures have also been determined. The establishment of subtle interactions among the different groups of plant hormones, and plant growth regulators operating either directly or indirectly, alone or in synergistic combination, have given to some degree an insight into certain interrelationships that may exist among cells, tissues and organs. The information is however by no means complete.
For some time it has been known that plant cell cultures can be maintained in a non-differentiating proliferative state indefinitely. It has, however, only been recently found that redifferentiation of tissues, organs or whole plant organisms can be experimentally induced. Since the demonstrations by Skoog et al. "Chemical regulation of growth and organ formation in plant tissues cultured in vitro" Symp. Soc. Exp. Biol. 11 18-130 (1958), incorporated herein by reference! that the relative ratio of a cytokinin to an auxin determines the nature of organogenesis in tobacco pith tissue. Reorganization or regeneration from callus cultures includes the formation of shoot primordia or embryos, both of which ultimately lead to plantlet development in vitro.
The tendency for organogenesis vs. embryogenesis still depends upon the species involved and the presence of certain triggering factors which are chemical and/or physical in nature.
In 1902, Haberlandt "Kulturversuche mit isolierten pflanzenzellen," Mat. KI. Kais. Akad. Wiss. Wien 111 62, incorporated herein by reference! postulated that plant cells possessed the ability to produce entire plants and predicted that this would someday be demonstrable in cell cultures. In 1965, Reinert "Untersuchungen uber die morphogenese an Gewebekulturen," Ber. dt. Bot. Ges. 71 15! and Steward et al. "Growth and organized development of cultured cells/II. Organization in cultures grown from freely suspended cells," Am. J. Bot. 45 705-708! working independently, confirmed the occurrence of in vitro somatic embryogenesis. (Both references are incorporated herein by reference.) In experimentally manipulating somatic embryogenesis it is believed that two components of the culture media, an auxin and the nitrogen source, play crucial roles.
It has also been shown that the process of somatic embryogenesis takes place in two stages: first, the induction of cells with embryogenic competence in the presence of a high concentration of auxin; and second, the development of embryonic cell masses into embryos in the absence of or at a low concentration of auxin.
The induction of organogenesis or embryogenesis leads to distinct structural patterns in the callus. Detailed study of several plant species has enabled certain generalizations to be made about the developmental pathways leading to shoot, bud or embryo development.
The application of tissue culture techniques to the regeneration of plants via organogenesis or embryogenesis remains perhaps the most important contribution of basic studies in morphogenesis to commercial application.
Beasley reported the formation of callus in ovule cultures of cotton in 1971 "In vitro culture of fertilized cotton ovules," Bioscience 21 906-907 (1971), incorporated herein by reference!. Later, Hsu et al. "Callus induction by (2-chlorethyl) phosphoric (CPA) acid in cultured cotton ovules," Physiol. Plant 36 150-153 (1976), incorporated herein by reference! observed a stimulation of growth of calli obtained from ovules due to the addition of CPA and gibberellic acid to the medium. Callus cultures from other explants such as (a) leaf Davis et al. "In vitro culture of callus tissues and cell suspensions from okra (Hibiscus esculentus) and cotton (Gossypium hirsutum)", "In vitro 9 395-398 (1974)", both incorporated herein by reference! (b) hypocotyl Schenk et al. "Medium and technique for induction and growth of monocotyledonous and dicotyledonous plant cell cultures," Can. J. Bot. 50 199-204 (1972), incorporated herein by reference! and (c) cotyledons Rani et al. "Establishment of Tissue Cultures of Cotton," Plant Sci. Lett. 7 163-169 (1976), incorporated herein by reference! have been established for Gossypium hirsutum and G. arboreum.
Katterman et al. "The influence of a strong reducing agent upon initiation of callus from the germinating seedlings of Gossypium barbadense," Physiol. Plant 40 98-101 (1977), incorporated herein by reference! observed that the compact callus from cotyledons of G. barbadense formed roots, and in one instance regeneration of a complete plant was also obtained. Smith et al. "Defined conditions for the initiation and growth of cotton callus in vitro, Gossypium arboreum," In vitro 13 329-334 (1977), incorporated herein by reference! determined conditions for initiation and subculture of hypocotyl-derived callus of G. arboreum. Subsequently, Price et al. "Callus cultures of six species of cotton (Gossypium L) on defined media," Pl. Sci. Lett. 8 115-119 (1977), and "Tissue culture of Gossypium species and its potential in cotton genetics and crop improvement," Beltwide Cotton Production Research Conference Proc. pp. 51-55 (1977), of the National Cotton Council, Memphis, each incorporated herein by reference! defined conditions for the initiation and subculture of callus from five species of Gossypium.
One of the common problems in establishing cultures of many plant species is the "browning" of the explant in the culture medium. In cotton, this leaching of polyphenols was overcome by replacing sucrose with glucose, and by transferring the cultures to a fresh medium every 10 days. After 3 or 4 passages on glucose supplemented medium, the browning completely disappeared and the cultures could be transferred back to sucrose-supplemented media. Although difficulties with the induction, browning and maintenance of calli during subcultures have been overcome with certain Gossypium species, all attempts to regenerate plants from callus cultures have been either unsuccessful or have involved several time-consuming steps. Davidonis et al. "Plant Regeneration from Callus Tissue of Gossypium hirsutum," L. Plant Sci. Lett. 32 89-93 (1983), incorporated herein by reference! reported the eventual formation of embryos two years after the initiation of culture.
Although many growth substances, such as natural phytohormones and synthetic growth regulating compounds have been utilized in tissue culture media to bring about plant regeneration in vitro, no generalization, much less specifics, of the effects of different substances on plant regeneration has been arrived at. Indeed, the same substances, when applied to different plant species, may either inhibit growth, enhance growth, or have no effect whatsoever. Therefore, aside from certain standard procedures, it remains necessarily a difficult task to arrive at a working protocol for plant regeneration for any new species and by many orders of magnitude a more difficult task to achieve plant transformation.
The present invention provides a method for the rapid regeneration of cotton plants from segments excised from seedlings. The method described offers a high degree of repeatability and reliability and it enables genetic transformation of cotton plants.